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KKU Journal of Basic and Applied Sciences
Journal homepage: jbas.kku.edu.sa Vol 4, 1 (2018) 9-13
P a g e | 9
Molecular Diversity Among Five Ferns Growing in Al-Souda Mountains, Abha Region, Kingdom of Saudi Arabia
Abdulrahman Alshehri
aand Mahmoud Moustafa
a,baBiology Department, Faculty of Science, King Khalid University
a,bBotany Department, Faculty of Science, South Valley University, Qena, Egypt.
Keywords: Ferns, RAPD, ISSR, Biomarkers
1.INTRODUCTION
Due to the small size of ferns plant many researchers neglected the extensive study of them. It could be found and live under a variety of environmental conditions including, wetlands, terrestrial and on rocks habitats whereas humid, rainfall and shade are available.
Some regions of Saudi Arabia have been studied for ferns flora especially area which have a comparatively high rainfall and humidity such as Aseer region other than arid regions such as the north-western and northern regions. For example, Migahid [1] , Collenette [2, 3] and Al-Turki [4] recorded two species of Selaginella, namely Selaginella yemensis (Swart) and Selaginella imbricata (Forssk). Al-Sheri [5], collected nine species including Adiantum capillus-veneris, Adiantium nigrium, Asplenium ceterach, Asplenium viridie, Asplenium aethiopicum, Asplenium filare, Asplenium trichomanes and Cheilanthes pteridioides and studied their abundance and some of them identified as a new record for the area. Kurschner [6], Basahy [7] and Al-Turki [4] showed some record about pteridophytes, flora in Aseer region, Saudi Arabia. Also Al- Sheri and Lashin [8] described two species Selaginella imbricata and Selaginella yemensis collected from El-Baha
region and Shammaran and Muhayl valley of Asir regionSaudi Arabia .
Tharwat Mountains especially in Aseer region, Saudi Arabia characterized by unique environmental conditions due to its location about 2500 meters above the sea level. Moreover, El- Souda Mountain in Aseer region has an elevation of 2700 meters above the sea level so why this region holds a variety of plants belongs to the ferns that is rarely found in any other region of Kingdom of Saudi Arabia. Most plant species evolve special morphological, ecological, and physiological adaptations to survive under special environmental conditions [9,10]. At the beginning of the taxonomical science, morphological characters have been used mainly to identify species, families and varieties. Recently, an application of molecular basis became widely accepted techniques for characterization of plant species up to varieties and sub- varieties level. In addition, there were little information about molecular characterization for most fern plants growing in Aseer region, KSA .
Therefore, in this research articles, molecular techniques were used to sort among five ferns grown naturally in Al-Souda Mountain, KSA namely Asplenium ceterach, Asplenium aethiopicum, Asplenium trichomanes, Cheilanthes Received:23 April 2018 / Revised: 21 July 2018 / Accepted: 19 September 2018 / Published: 25 December 2018
Abstract: In this study, five samples from ferns plants namely Asplenium ceterach, Asplenium aethiopicum, Asplenium trichomanes, Cheilanthes pteridioides, Adiantum capillus-veneris were collected from El Souda Mountains, Abha area, Saudi Arabia. The DNA was extracted from fresh leaves and five random amplified polymorphic DNA (RAPD) markers, five inter-simple sequence repeat (ISSR) markers were used to investigate the genetic variations of the investigated plants. ISSR markers produced 39.0 bands recording 7.5 bands per marker with a polymorphism 60.57% and the highest genetic similarity (82.05%) was found between A. ceterach and A. aethiopicum, while the lowest similarity (28.21%) between A. trichomanes and C. pteridioides. RAPD markers produced 45.0 bands with a polymorphism 77.81% and the highest similarity (61.70%) was between A. aethiopicum and A. ceterach and also between C. pteridioides and A. capillus-veneris while the lowest similarity value (36.71%) between A. capillus-veneris and A. aethiopicum. Summation ISSR, RAPD primers provided the highest similarity value (70.93%) that was found between A. aethiopicum and A. trichomanes, while the lowest similarity value (34.88%) was between C. pteridioides and A. trichomanes. In conclusion, applied biomarkers including RAPD, ISSR showed varied capability to distinguish among the genotypes of ferns plants.
pteridioides and Adiantum capillus-veneris to overcome the limitations of morphological characterization methods.
Random Amplified Polymorphic DNA (RAPD) markers and Inter Simple Sequence Repeat (ISSR) have been used for studying diversification.
2.MATERIALSANDMETHODS
Sampling localities
The surveyed area has an elevation bout 2851meters, latitude 18.28952 and longititude 42.36329 and the plant samples collected between March 15, 2016 to June 14, 2016.
Determination of lichens were done by Prof. Dr. Abdel Rhaman Mohei El shehri, Biology Dep. Faculty of Science, King Khalid University, KSA in accordance with Al-Shehri [2] and Phillips [11]. The sample of lichens are kept in the herbarium of Biology Dep. Faculty of Science, King Khalid University.
Plant material
Five ferns plants namely A. ceterach, A. aethiopicum, A.
trichomanes, C. pteridioides and A. capillus-veneris have been collected from the surveyed area. The sampled leaves from each plant was immediately placed in a sealed plastic bag then in dry ice for the DNA extraction.
Extraction the DNA from leaves
The DNA was extracted from leaves by DNA extractions kit in fourteen steps as followings: 1) 0.90 grams from fresh leaves was grinded to a fine powder using liquid nitrogen by a mortar and pestle. 2) 400 μl and 4 μl of RNase from AP1were added then vortexed vigorously for 1 min. 3) Samples were incubated for 10 min at 65°C 4) 130 μl from Buffer AP2 was added and incubated for 5 min on ice 5) The lysate was centrifuged for 5 min at 20.000 xg. 6) The supernatant was applied to QIAshredder spin column and placed in a 2 ml collection tube 7) Samples were subjected to centrifugation for 2 min at 20.000 xg. 8) The flow-through was placed into a new tube collection tube. 9) 900 ul of AW1 was added and mixed by pipetting 10) 650 ul was transferred into a DNeasy Mini spin column 11) Samples were subjected to centrifugation for 1 min at 7000 xg. 12) The flow through was discard, then the spin column was placed into a new 2 ml collection tube. 13) 500 ml from buffer AW2 was added then centrifuged for 1 min at 7000 xg and this step repeated two times 14) The spin column was transferred to a new 1.5 ml. tube 14) 100 ul of Buffer AE for elution was added.
Estimation of DNA concentration and Molecular weight markers
The concentration of extracted genomic DNA was measured by using a Thermo Scientific™ BioMate 3S UV-Visible at 260 nm and 1 kb DNA ladder was used.
Agarose gel electrophoresis and biomarkers
10 RAPDs and ISSRs markers were used in final concentration of 9 pmol/μl. The primers applied and their sequences are listed in (Table 1). By using GoTaq Green Master Mix, the standard PCRs buffer were performed. 1X GoTaq Green Master Mix, 19 pmol of primer, 23 ng genomic DNA and nuclease free water to obtains a final volume of 25 µl have been used as described previously [12]. A negative control reaction without the DNA template have been applied.
PCR program
Cycling condition as follows: Initial denaturartion at 94°C for 5 min followed by 39 cycles of denaturation at 94°C for 1 min, annealing temperature 31 °C for 1 min, extension at 72°C for 2 min and final extension at 72°C for 7 minutes by PTC 200 Peltier Thermal Cycler (MJ Research — USA). As described previously by Moustafa et al., [12, 13], Hashemi et al., [14], stained 1.43% of agarose gels with ethidium bromide was used for visualization of DNA fragments. 19 μl of PCR amplified products were applied and each gel that run horizontally in 0.5X Tris-borate-EDTA (TBE) buffer. DNA amplified bands were visualized by UV transilluminator.
Data analysis
Scored RAPD and ISSR amplified bands were manually counted as present (1) or absent (0). Squared euclidean distance and Ward’s method of Statistics version 5.0 a computer was used to calculate the distances and to generate dendrogram. The amount of polymorphism among tested plant was evaluated [14, 15].
3.RESULTSANDDISCUSSION
The genotypes of five ferns were studied from Al Souda Mountains, Saudi Arabia using numbers of biomarkers for the first time. It was found that the genotypes are different totally among the studied ferns plants. In addition, the results showed that RAPD, ISSR and the sum of them used in this study could effectively used in determining the differences among the different plants genotypes. The genetic similarity coefficients based on ISSR marker showed a polymorphism 60.75% and the highest genetic similarity (82.05%) was found between A.
ceterach and A. aethiopicum, followed by A. ceterach and A.
trichomanes (64.10%) while the lowest similarity index (28.21%) between C. pteridioides and A. trichomanes (Fig. 1 and Table I). Obtained dendrogram based one eluciden output grouped the five genotypes int0o three main clusters at 8.83 linkage distance. The first cluster comprised three genotypes including A. ceterach, A. aethiopicum and A. trichomanes. A.
trichomanes separated from A. ceterach and A. aethiopicum while A. ceterach and A. aethiopicum formed one clusters at 3.5 linkage. C. pteridioides and Adiantum capillus-veneris found to have in one calde at linkage distance of 8.00. The five ISSR biomarkers yielded 39.0 bands forming 7.5 per primer, out of which 24.0 were found to be polymorphic, 4.0 monomorphic and 10 unique bands (Table I, II and Fig. 1).
The genetic similarity coefficients based on RAPD marker showed 45.0 bands with 7.0 per primer and revealed that the highest similarity (72.34%) was between A. aethiopicum and A. trichomanes and between C. pteridioides and Adiantum capillus-veneris (61.70 %) followed by C. pteridioides and A.
aethiopicum (53.19%) while the lowest similarity value (36.71%) between A. capillus-veneris and A. aethiopicum. The polymorphism rate found to be 77.81% and the recorded monomorphic bands and 10 unique bands. Obtained dendrogram depending on eluciden output grouped the five genotypes into three main clusters at 16.3 linkage distance. A.
ceterach and A. aethiopicum formed one +cluster and the second one including A. trichomanes and C. pteridioides while Adiantum capillus-veneris fromed one calde at linkage distance of 18.6 (Table I, III and Fig. 1).
Summation the total genetic similarity coefficients based on RAPD and ISSR biomarkers showed that the highest similarity value (70.93%) was between A. aethiopicum and A. ceterach followed by A. trichomanes and A. ceterach (60.47%) and the same similarity rate found also between C. pteridioides and A.
capillus-veneris (60.47%). The lowest similarity value (34.88%) was between C. pteridioides and A. trichomanes.
Obtained dendrogram grouped the five genotypes of into three main clusters at 24.5 linkage distance. The first cluster found to have three genotypes including A. ceterach, A. aethiopicum in one clade whereas Asplenium trichomanes separated from them. As in ISSR A. capillus-veneris and C. pteridioides found to be in one clade (Table I, IV and Fig. 1).
Table I: Polymorphism of 10 ISSR and RAPD biomarkers applied on five ferrns plants
Primer sequence Bands No Polymorphic Bands
Monomorphic Bands
Unique Bands
Polymorphis m %
5'- ACACACACACA CAAG-3 8.00 4.00 0.00 4.00 50.00
5'- TCTCTCTCTCTC TCTCC-3 6.00 1.00 2.00 3.00 16.67
5'-CACACACACAC ACACC-3 8.00 5.00 0.00 3.00 62.50
5'-GTGTGTGTGTG TCC-3 7.00 6.00 1.00 0.00 85.71
5'-AGAGAGAGAG AGAGC-3 9.00 8.00 1.00 0.00 88.89
Total ISSR 39.0 24.0 4.00 10.00 Average 60.75
5'-CACGGCGAT-3' 8.00 6.00 0.00 2.00 75.00
5'-GAGATCCCTC-3 10.0 7.00 0.00 3.00 70.00
5'-GGAGCCCCCT-3 11.0 10.00 0.00 1.00 90.91
5'-GAACACTGGG-3 8.00 6.00 0.00 2.00 75.00
5'-GAGATCCCTC-3 8.00 6.00 0.00 2.00 75.00
Total RAPD 45.0 35.0 0.00 10.0 Average 77.18
Fig. 1: Dendrogram depicting the genetic relationship among 5 ferns plants
genotypes growing in El Soda Mountaisn, KSA, based on ISSR, RAPD and ISSR+RAPD data
Table II: Similarity index based on ISSR biomarkers A.
Ceterach
A.
Aethiopicum
A.
Trichomanes
C.
Pteridioides
A.
Capillus- veneris A. ceterach 100
A. aethiopicum 0.8205 100
A. trichomanes 0.641 0.5128 100
C. pteridioides 0.5897 0.5641 0.2821 100
A. capillus-veneris 0.4359 0.4615 0.4872 0.5897 100
Table III: Similarity index based on RAPD biomarkers A.
Ceterach
A.
Aethiopicum
A.
Trichomanes
C.
Pteridioides
A.
Capillus- veneris A. ceterach 100
A. aethiopicum 0.617 100
A. trichomanes 0.5745 0.4468 100
C. pteridioides 0.4468 0.5319 0.4043 100
A. capillus-veneris 0.4043 0.3617 0.4468 0.617 100
Table IV: Similarity index based on RAPD + ISSR biomarkers A.
Ceterach
A.
Aethiopicum
A.
Trichomanes
C.
Pteridioides
A.
Capillus- veneris
A. ceterach 100
A. aethiopicum 70. 93 100
A. trichomanes 0.6047 0.4767 100
C. pteridioides 0.5116 0.5465 0.3488 100
A. capillus-veneris 0.4186 0.407 0.4651 0.6047 100
Recent development and application of molecular biology techniques has been achieved for the evaluation the extent of genetic diversity and population genetic structure. It is evident that different biomarkers have different identification capability that reflect different aspects of molecular characterizations.
The results obtained here are to some extent in consistent to the other study found by many researchers. For example, two ferns by RAPDs markers namely Pyrrosia piloselloides and Pyrrosia longifolia had been analyzed and found that there were various rate of polymorphism and the numbers of amplified bands ranged from 2 to 14 bands [16]. The similarity index matrix showed that populations had 67 % to 84 % with different length/width ration. However, Ceratopteris thalictroides (Pteridaceae) had been collected from five area in China and examined by random amplification of polymorphic DNA (RAPD) and intersimple sequence repeat (ISSR) markers to evaluate the genetic diversity within and among 13 populations found that the percentages rate of polymorphic bands were 61%
and 65% for RAPD ISSR respectively [17]. In addition the
genetic diversity obviously varied among populations, with the percentages rate of polymorphic bands in range between 15% to 39% for RAPD and between 20% to 39% for ISSR.
Hamrick and Godt [18] showed that most plant species with wide distributions have high genetic diversity. It was explained that many rare and endangered plant species may have reduced their genetic diversity because of their small population size, and the plant species probably differentiated into genetically unique populations adapted to local environmental conditions for growth and survival [19, 20, 21]. Chalmers et al., [22] studied the genetic variability between Gliricidia sepium and G. maculate using RAPD markers and found that there were extensive genetic differences and the differences was higher between populations than within populations.
RAPD and ISSR markers have been used extensively to determine the genetic diversity among plant species. Recent study showed that RAPD and ISSR markers were used to determine the genetic diversity and phylogenetic relationships in several plant species, for example, Ziziphus spina-christi, Euphorbia spp, Phoenix dactylifera, Schinus
P a g e | 13 molle [12, 13, 14, 23]. Sobotka et al., [24] evaluated the
polymorphism rate of Brassica napus and showed that polymorphic bands number range between 31% and 76%.
Also Williams et al., [25] reported that this variation in polymorphisms rate might be due to the deletion, addition or substitution of base within the priming site sequence.
Such differences between the two marker to detect variation in A. ceterach, A. aethiopicum, A. trichomanes, C.
pteridioides, A. capillus-veneris may be due to genomic sequence diversity, number of alleles per locus/or loci and their distribution within each plant species differ from each others.
4.CONCLUSION
Overall, our finding showed that both DNA markers including RAPD and ISSR applied to the five ferns species exhibited varied level of polymorphism and should be used in future work to determine the genetic diversity among ferns species.
5.ACKNOWLEDGMENT
Authors are thankful to the King Khalid University Abha, Saudi Arabia for the financial support under of this research article.
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